Mussel byssal threads are an extraordinary type of acellular connective tissue. Composed of collagen, they are made in minutes, resist degradation when protected by a cuticle, and have a range of desirable mechanical properties that resemble those of important biopolymers such as tendon collagen, elastin, and dragline silk. The long range objective of this research has been to discover how the mechanical properties of byssus are determined by its molecular architecture. Specifically, the proposed research attempts to discover what effect the distribution of three naturally occurring fusion proteins in byssal threads has on the ability of these structures to recover both initial length and modulus after yield.The proteins, preCol-NG, preCol-P and preCol-D, are all characterized by a block copolymer structure that consists of a central collagenous domain sandwiched between two flanking domains, and capped by histidine-rich amino- and carboxy-termini. The flanking domains resemble elastin (preCol-P) and spider dragline silk (preCol-D). The major aims (methods) of this proposal are to determine a) how preCols are oriented and aligned during fiber assembly (using reversible bifunctional crosslinkers), b) what effect maturation of byssus has on N- and C-terminal Dopa residues and on disulfide formation in preCols (phenylhydrazine derivatization for Dopa, followed by limited digestion and MALDI TOF analysis), c) whether transition metals function to cross-link preCols through their histidine-rich termini (metal removal by chelation followed by micromechanical analysis);, and d) whether individual preCol molecules can be stretched on an atomic force microscope. The outcome of these should be of particular relevance to the design of scaffolding biomaterials with a wide range of mechanical properties.